Table of Contents |
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Overall Workflow
- A stack of serial tSEM images (a complete set of original images with optimized focus, brightness, and contrast; Don't forget to take an image of a calibration grid!)
- Image alignment with Fiji/TrakEM2
- Stack-crop the aligned images, export as .tif files
- Assign a series code and rename image files
- Get a spreadsheet ready for series data collection
- Import aligned images into RECONSTRUCT
- Manually fine-tune alignment, if necessary
- Calibration: x-y (pixel size) with a calibration grid image; z (section thickness) by the cylindrical mitochondria method
- Choose and trace neuropil elements (and organelles) of interest
- Data analysis
- Publication, etc.
Things to do before Reconstructing:
!IMPORTANT! Make sure you have access to TACC (John is the current contact person).
Pre-processing of serial section EM images before importing into RECONSTRUCT:
- Alignment of serial section EM images using Fiji/TrakEM2:
- old protocol for TEM images
- new protocol for tSEM images using TACC environment
- Assign a series code (Harris lab specific)
- Rename aligned serial EM image files with Bulk Rename Utility
- Suggested format: XXXXX_### (XXXXX = series code; ### = section number; 000 = calibration grid image)
Protocols for Reconstruct (and other analysis tools):
- Get your Reconstruct here! (Use 32-bit version; 64-bit may be buggy)
- Instructions for new users of Reconstruct: Reconstruct: Intro for Beginners, Reconstruct Basics, User Manual.
- Starting a New Series
- Section Thickness
- Calibration Error Correction Strategy
- Adjusting brightness/contrast
- Editing Trace Pallettes
- Importing Traces
- Unbiased 3D stereology
- Dendrites:
- Counting/tracing mictotubules
- Tracing Z-Lengths
- Tracing plinn (linear nearest-neighbor distance between protrusions)
- Spines:
- Stamping Dendritic Protrusions
- Cutting off spines and spine heads: Reconstruct / Blender (with Python add-on described in Bartol et al., 2016)
- Synapses:
- Identifying Symmetric and Asymmetric Synapses
- Tracing synapses (nascent and active zones)
- Axons:
- Organelles:
- Ribosome Identification
- Stamping and tracing smooth endoplasmic reticulum and spine apparatus
- Exporting quantitative data
- RECONSTRUCT color scheme
- pyRECONSTRUCT merge tool
- CellBlender: Github | MCell and CellBlender | Tutorial
- NeuroMorph: http://cvlab.epfl.ch/NeuroMorph
Data Analysis Protocols:
- Data Entry and Dendrite Analysis Spreadsheet
- Statistical Analyses with STATISTICA
- Making Box and Whisker Plots in Excel
- Data Transforms
- Statistics for Biologists (Nature collection)